PROJECT SUMMARY Bacteria assemble large extracellular complexes called nanomachines to differentially interact with their environment. Nanomachines enact specific functions, including substrate secretion, cell motility, and pathogenesis. The regulation and structural composition of bacterial protein nanomachines is inherently complex. One such nanomachine, the flagellar apparatus, is critical for bacterial motility, and its assembly is highly ordered. Many regulatory systems are in place to ensure that proper assembly of the flagella occurs both spatially and temporally. Many studies investigate the composition of the flagellar structure as well as how the host regulates transcription and translation of its various components. However, less is known regarding the systems in place controlling accurate assembly of the flagellum. One of the major components of the flagellum is the extracellular filament, which is responsible for generating thrust to mobilize the cell. The majority of studies focusing on filament assembly use the Gram- negative organisms Salmonella typhimurium and Escherichia coli. Investigations suggest that these two closely related organisms maintain distinct mechanisms regulating filament length, as well as repair. Whether the precise regulation of filament length is necessary for efficient motility across all species is unknown. Using the genetically tractable, Gram-positive model bacterium Bacillus subtilis, we propose to determine the mechanisms employed by this organism to control filament growth and length, as well as whether filament repair occurs. Additionally, mechanisms regulating the length of the flagellar rod spanning from the membrane-bound flagellar motor to the extracellular filament in B. subtilis is unknown. Four proteins putatively make up the B. subtilis rod, but their order of assembly is currently unknown. Further, the rod must precisely traverse a large distance in both Gram-negative (periplasm) and Gram-positive (peptidoglycan) organisms to initiate assembly of the flagellar filament. Thus, the cell must regulate rod length to accurately span these depths. Although studies in the Gram-negative S. typhimurium show rod length is determined via interaction with the outer membrane, a lack of an outer membrane in Gram-positive organisms indicates a different mechanism is in place. We will identify the structural composition of the rod and determine the components controlling rod length. The aims of this proposal and experiments suggested focus on the structural organization of the flagellar rod and filament, specifically to: (i) assess the cell mechanisms controlling flagellar filament length and elongation, and (ii) define the components that make up the rod and determine the regulation of its length and assembly. We will address the proposed experiments using genetic, biochemical, and cell biology techniques. Overall, these aims intend to promote our understanding of bacterial nanomachine regulation and their contributions to cell physiology and fitness.